Glucagon analogs exhibiting enhanced solubility in physiological pH buffers

ABSTRACT

Modified glucagon peptides are disclosed having improved solubility while retaining glucagon agonist activity. The glycogen peptides have been modified by substitution of native amino acids with, and/or addition of, charged amino acids to the carboxy terminus of the peptide. The modified glucagon agonists can be further modified by pegylation, or the addition of a carboxy terminal peptide selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, or both to further enhance the solubility of the glucagon agonist analogs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national counterpart application ofinternational application serial No. PCT/US2008/050099 filed Jan. 3,2008, which claims priority to U.S. Provisional Patent Application No.60/878,919, filed Jan. 5, 2007. The entire disclosures ofPCT/US2008/050099 and U.S. Ser. No. 60/878,919 are hereby incorporatedby reference.

BACKGROUND

Hypoglycemia occurs when blood glucose levels drops too low to provideenough energy for the body's activities. In adults or children olderthan 10 years, hypoglycemia is uncommon except as a side effect ofdiabetes treatment, but it can result from other medications ordiseases, hormone or enzyme deficiencies, or tumors. When blood glucosebegins to fall, glucagon, a hormone produced by the pancreas, signalsthe liver to break down glycogen and release glucose, causing bloodglucose levels to rise toward a normal level. However for diabetics,this glucagon response to hypoglycemia may be impaired, making it harderfor glucose levels to return to the normal range.

Hypoglycemia is a life threatening event that requires immediate medicalattention. The administration of glucagon is an established medicationfor treating acute hypoglycemia and it can restore normal levels ofglucose within minutes of administration. When glucagon is used in theacute medical treatment of hypoglycemia, a crystalline form of glucagonis solubilized with a dilute acid buffer and the solution is injectedintramuscularly. While this treatment is effective, the methodology iscumbersome and dangerous for someone that is semi-conscious.Accordingly, there is a need for a glucagon analog that maintains thebiological performance of the parent molecule but is sufficientlysoluble and stable, under relevant physiological conditions, that it canbe pre-formulated as a solution, ready for injection.

Additionally, diabetics are encouraged to maintain near normal bloodglucose levels to delay or prevent microvascular complications.Achievement of this goal usually requires intensive insulin therapy. Instriving to achieve this goal, physicians have encountered a substantialincrease in the frequency and severity of hypoglycemia in their diabeticpatients. Accordingly, improved pharmaceuticals and methodologies areneeded for treating diabetes that are less likely to induce hypoglycemiathan current insulin therapies.

As described herein, high potency glucagon agonists are provided thatexhibit enhanced biophysical stability and aqueous solubility atphysiological pH in pharmaceutical compositions suitable for commercialuse. Native glucagon is neither soluble, nor stable in the physiologicalpH range and thus must be manufactured as a dry product that requiresreconstitution and immediate use. The glucagon analogs described hereinhave enhanced physical properties that render them superior for use incurrent medicinal settings where the native hormone is currentlyemployed. These compounds can be used in accordance with one embodimentto prepare pre-formulated solutions ready for injection to treathypoglycemia. Alternatively, the glucagon agonists can beco-administered with insulin to buffer the effects of insulin to allowfor a more stable maintenance of blood glucose levels. In addition,other beneficial uses of compositions comprising the modified glucagonpeptides disclosed herein are described in detail below.

SUMMARY

One embodiment of the invention provides glucagon peptides that retainglucagon receptor activity and exhibit improved solubility relative tothe native glucagon peptide (SEQ ID NO: 1). Native glucagon exhibitspoor solubility in aqueous solution, particularly at physiological pH,with a tendency to aggregate and precipitate over time. In contrast, theglucagon peptides of one embodiment of the invention exhibit at least2-fold, 5-fold, or even higher solubility compared to native glucagon ata pH between 6 and 8, for example, at pH 7 after 24 hours at 25° C.

In one embodiment the glucagon peptides retain at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 75% activity, 80% activity, 85% activity, or 90% ofthe activity of native glucagon. In one embodiment, the glucagonpeptides of the present invention have potency greater than glucagon.Any of the glucagon peptides of the invention may additionally exhibitimproved stability and/or reduced degradation, for example, retaining atleast 95% of the original peptide after 24 hours at 25° C.

In accordance with one embodiment a glucagon peptide is provided whereinthe peptide is modified by amino acid substitutions and/or additionsthat introduce a charged amino acid into the C-terminal portion of thepeptide, and in one embodiment at a position C-terminal to position 27of SEQ ID NO: 1. Optionally, one, two or three charged amino acids maybe introduced within the C-terminal portion, and in one embodimentC-terminal to position 27. In accordance with one embodiment the nativeamino acid(s) at positions 28 and/or 29 are substituted with a chargedamino acid, and/or one to three charged amino acids are added to theC-terminus of the peptide. In exemplary embodiments, one, two or all ofthe charged amino acids are negatively charged. Additionalmodifications, e.g. conservative substitutions, may be made to theglucagon peptide that still allow it to retain glucagon activity.

In accordance with one exemplary embodiment the glucagon peptidecomprises an amino acid sequence of SEQ ID NO: 11, or an analog thereofthat contains 1 to 3 further amino acid modifications relative to nativeglucagon, or a glucagon agonist analog thereof. SEQ ID NO: 11 representsa modified glucagon peptide wherein the asparagine residue at position28 of the native protein has been substituted with an aspartic acid. Inanother exemplary embodiment the glucagon peptide comprises an aminoacid sequence of SEQ ID NO: 38, wherein the asparagine residue atposition 28 of the native protein has been substituted with glutamicacid. Other exemplary embodiments include glucagon peptides of SEQ IDNOS: 24, 25, 26, 33, 35, 36 and 37.

The solubility of any of the foregoing compounds can be further improvedby attaching a hydrophilic moiety to the peptide. In one embodiment thehydrophilic moiety is a polyethylene glycol chain or other water solublepolymer that is covalently linked to the side chain of an amino acidresidue at position 16, 17, 21 or 24 of said glucagon peptide. Thepolyethylene glycol chain in accordance with one embodiment has amolecular weight selected from the range of about 500 to about 40,000Daltons. The present invention further encompasses pharmaceuticallyacceptable salts of said glucagon agonists.

In other exemplary embodiments, any of the foregoing compounds can befurther modified to alter its pharmaceutical properties by the additionof a second peptide to the carboxy terminus of the glucagon peptide. Inone embodiment a glucagon peptide is covalently bound through a peptidebond to a second peptide, wherein the second peptide comprises asequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO:21 and SEQ ID NO: 22.

In yet further exemplary embodiments, any of the foregoing compounds canbe further modified to improve stability by modifying the amino acid atposition 15 of SEQ ID NO: 1 to reduce degradation of the peptide overtime, especially in acidic or alkaline buffers.

In accordance with one embodiment a pharmaceutical composition isprovided comprising any of the novel glucagon peptides disclosed herein,preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrieror excipient. Such compositions may contain a glucagon peptide at aconcentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml,5 mg/ml, or higher. In one embodiment the pharmaceutical compositionscomprise aqueous solutions that are sterilized and optionally storedwithin various containers. In other embodiments the pharmaceuticalcompositions comprise a lyophilized powder. The pharmaceuticalcompositions can be further packaged as part of a kit that includes adisposable device for administering the composition to a patient. Thecontainers or kits may be labeled for storage at ambient roomtemperature or at refrigerated temperature.

In accordance with one embodiment a method of rapidly increasing glucoselevel or treating hypoglycemia using a pre-formulated aqueouscomposition is provided. The method comprises the step of administeringan effective amount of an aqueous solution comprising a novel modifiedglucagon peptide of the present disclosure. In another embodiment amethod is provided for inducing the temporary paralysis of theintestinal tract. The method comprises the step of administering one ormore of the glucagon peptides disclosed herein to a patient in needthereof.

In yet another embodiment a method of reducing weight gain or inducingweight loss is provided, which involves administering an effectiveamount of an aqueous solution comprising a glucagon peptide of theinvention. In further embodiments, methods of treating diabetesinvolving co-administering insulin and a glucagon peptide of theinvention are provided.

Exemplary glucagon peptides are selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11and SEQ ID NO: 33, wherein amino acid 29 of the glucagon peptide isbound to a second peptide through a peptide bond, and said secondpeptide comprises the sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ IDNO: 22. In one embodiment the glucagon peptide is pegylated. In oneembodiment the method comprises the step of administering a peptidecomprising the sequence of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO:26, wherein a polyethylene chain is covalently linked to amino acidposition 21 or at position 24.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph representing the stability of GlucagonCys²¹maleimidoPEG_(5K) at 37° C. incubated for 24, 48, 72, 96, 144 and166 hours, respectively.

FIG. 2 represents data generated from HPLC analysis of GlucagonCys²¹maleimidoPEG_(5K) at pH 5 incubated at 37° C. for 24, 72 or 144hours, respectively.

FIG. 3 represents data showing the solubility of glucagon analogs (D28,E29, E30) relative to native glucagon after 60 hours at 25° C. at pH of2, 4, 5.5, 7 and 8, respectively.

FIG. 4 represents data showing the solubility of glucagon analogs(E15D28, D28E29 and D28E30) relative to native glucagon after 24 hoursat 25° C. and then 24 hours a 4° C. at pH of 2, 4, 5.5 and 7,respectively.

FIG. 5 represents the maximum solubility of glucagon analogs D28, D28E30and E15, D28 after 24 hours, pH 7 at 4° C.

FIG. 6 represents data showing a glucagon receptor mediated cAMPinduction by glucagon analogs (K29 ▴, K30 ▾, and K29K30♦) relative tonative glucagon ▪.

FIG. 7 represents data showing a glucagon receptor mediated cAMPinduction by glucagon analogs (D28□, E29 Δ, E30 ∇, K30K31 ⋄ and K30, ▾)relative to native glucagon ▪.

FIG. 8 represents data showing a glucagon receptor mediated cAMPinduction by glucagon analogs (D28□, E28

and K29, ▴) relative to native glucagon ▪.

FIG. 9 represents data showing a glucagon receptor mediated cAMPinduction by glucagon analogs (D28E29+, D28E30 X, E15D28* and E29 Δ)relative to native glucagon ▪.

FIG. 10 represents data showing the change in serum glucoseconcentrations in beagle dogs after intramuscular administration ofglucagon and glucagon analogs. The animals were administered a 0.005mg/kg dose of either glucagon, a glucagon analog comprising glucagonwith the sequence of SEQ ID NO: 31 linked to the carboxy terminus ofglucagon (glucagon-CEX) or a glucagon analog comprising an aspartic acidsubstitution at amino acid 28 (glucagon-Asp28) SEQ ID NO: 11.

DETAILED DESCRIPTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia, asmeasured, for example, by an increase in blood glucose level. The amountthat is “effective” will vary from subject to subject, depending on theage and general condition of the individual, mode of administration, andthe like. Thus, it is not always possible to specify an exact “effectiveamount.” However, an appropriate “effective” amount in any individualcase may be determined by one of ordinary skill in the art using routineexperimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a faun that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment. As used herein, the term “purified” doesnot require absolute purity; rather, it is intended as a relativedefinition. The term “purified polypeptide” is used herein to describe apolypeptide which has been separated from other compounds including, butnot limited to nucleic acid molecules, lipids and carbohydrates.

The term “isolated” requires that the referenced material be removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotidepresent in a living animal is not isolated, but the same polynucleotide,separated from some or all of the coexisting materials in the naturalsystem, is isolated.

A “glucagon peptide” as used herein includes any peptide comprising,either the amino acid sequence of SEQ ID NO: 1, or any analog of theamino acid sequence of SEQ ID NO: 1, including amino acid substitutions,additions, or deletions, or post translational modifications (e.g.methylation, acylation, ubiquitination and the like) of the peptide,that stimulates glucagon or GLP-1 receptor activity, as measured by cAMPproduction using the assay described in Example 13.

The term “glucagon agonist” refers to a complex comprising a glucagonpeptide that stimulates glucagon receptor activity, as measured by cAMPproduction using the assay described in Example 13.

As used herein a “glucagon agonist analog” is a glucagon peptidecomprising a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NO: 13 or a analog of such a sequence that has beenmodified to include one or more conservative amino acid substitutions atpositions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27,28 or 29.

As used herein an amino acid “modification” refers to a substitution,addition or deletion of an amino acid, and includes substitution with oraddition of any of the 20 amino acids commonly found in human proteins,as well as atypical or non-naturally occurring amino acids. Throughoutthe application, all references to a particular amino acid position bynumber (e.g. position 28) refer to the amino acid at that position innative glucagon (SEQ ID NO:1) or the corresponding amino acid positionin any analogs thereof. For example, a reference herein to “position 28”would mean the corresponding position 27 for a glucagon analog in whichthe first amino acid of SEQ ID NO: 1 has been deleted. Similarly, areference herein to “position 28” would mean the corresponding position29 for a glucagon analog in which one amino acid has been added beforethe N-terminus of SEQ ID NO: 1.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

-   -   I. Small aliphatic, nonpolar or slightly polar residues:        -   Ala, Ser, Thr, Pro, Gly;    -   II. Polar, negatively charged residues and their amides:        -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;    -   III. Polar, positively charged residues:        -   His, Arg, Lys; Ornithine (Orn)    -   IV. Large, aliphatic, nonpolar residues:        -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine    -   V. Large, aromatic residues:        -   Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term “polyethylene glycol” or “PEG”, refersto mixtures of condensation polymers of ethylene oxide and water, in abranched or straight chain, represented by the general formulaH(OCH₂CH₂)_(n)OH, wherein n is at least 9. Absent any furthercharacterization, the term is intended to include polymers of ethyleneglycol with an average total molecular weight selected from the range of500 to 40,000 Daltons. “polyethylene glycol” or “PEG” is used incombination with a numeric suffix to indicate the approximate averagemolecular weight thereof. For example, PEG-5,000 refers to polyethyleneglycol having a total molecular weight average of about 5,000.

As used herein the term “pegylated” and like terms refers to a compoundthat has been modified from its native state by linking a polyethyleneglycol polymer to the compound. A “pegylated glucagon peptide” is aglucagon peptide that has a PEG chain covalently bound to the glucagonpeptide.

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini. For example, anamino acid chain comprising an amide group in place of the terminalcarboxylic acid is intended to be encompassed by an amino acid sequencedesignating the standard amino acids.

As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups.

As used herein a “dimer” is a complex comprising two subunits covalentlybound to one another via a linker. The term dimer, when used absent anyqualifying language, encompasses both homodimers and heterodimers. Ahomodimer comprises two identical subunits, whereas a heterodimercomprises two subunits that differ, although the two subunits aresubstantially similar to one another.

As used herein the term “pH stabilized glucagon peptide” refers to aglucagon agonist analog that exhibits superior stability and solubility,relative to native glucagon, in aqueous buffers in the broadest pH rangeused for pharmacological purposes.

As used herein the term “charged amino acid” refers to an amino acidthat comprises a side chain that is negatively charged (i.e.,de-protonated) or positively charged (i.e., protonated) in aqueoussolution at physiological pH. For example negatively charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positively charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 amino acids commonly found in human proteins,as well as atypical or non-naturally occurring amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety, including for example, a carboxylicacid or sulfonic acid group.

Embodiments

Applicants have discovered that native glucagon can be modified byintroducing charge at its carboxy terminus to enhance the solubility ofthe peptide while retaining the agonist properties of the peptide. Theenhanced solubility allows for the preparation and storage of glucagonsolutions at near neutral pH. Formulating glucagon solutions atrelatively neutral pHs (e.g. pH of about 6.0 to about 8.0) improves thelong term stability of the glucagon peptides. Accordingly, oneembodiment of the present invention is directed to a glucagon agonistthat has been modified relative to the wild type peptide ofHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 1) to improve the peptide's solubility in aqueous solutions,particularly at a pH ranging from about 5.5 to about 8.0, whileretaining the native peptide's biological activity. In one embodimentcharge is added to the peptide by the substitution of native non-chargedamino acids with charged amino acids selected from the group consistingof lysine, arginine, histidine, aspartic acid and glutamic acid, or bythe addition of charged amino acids to the amino or carboxy terminus ofthe peptide. Surprisingly, applicants have discovered that substitutingthe normally occurring amino acid at position 28 and/or 29 with chargedamino acids, and/or the addition of one to two charged amino acids atthe carboxy terminus of the glucagon peptide, enhances the solubilityand stability of the glucagon peptides in aqueous solutions atphysiologically relevant pHs (i.e., a pH of about 6.5 to about 7.5) byat least 5-fold and by as much as 30-fold.

Accordingly, glucagon peptides of one embodiment of the invention retainglucagon activity and exhibit at least 2-fold, 5-fold, 10-fold, 15-fold,25-fold, 30-fold or greater solubility relative to native glucagon at agiven pH between about 5.5 and 8, e.g., pH 7, when measured after 24hours at 25° C. Any of the glucagon peptides disclosed herein mayadditionally exhibit improved stability at a pH within the range of 5.5to 8, for example, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98%or 99% of the original peptide after 24 hours at 25° C. The glucagonpeptides may include additional modifications that alter itspharmaceutical properties, e.g. increased potency, prolonged half-lifein circulation, increased shelf-life, reduced precipitation oraggregation, and/or reduced degradation, e.g., reduced occurrence ofcleavage or chemical modification after storage.

In one embodiment a glucagon peptide with improved solubility may beprepared, for example, by introducing one, two, three or more chargedamino acid(s) to the C-terminal portion of native glucagon, and in oneembodiment at a position C-terminal to position 27. Such a charged aminoacid can be introduced, for example by substituting a native amino acidwith a charged amino acid, e.g. at positions 28 or 29, or alternativelyby adding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. In other embodiments, one, two, three or all ofthe charged amino acids are positively charged. In specific exemplaryembodiments, the glucagon peptide may comprise any one or two of thefollowing modifications: substitution of N28 with E; substitution of N28with D; substitution of T29 with D; substitution of T29 with E;insertion of E after position 27, 28 or 29; insertion of D afterposition 27, 28 or 29. For example, E28E30, D28E30

Additional modifications may be made to the glucagon peptide which mayfurther increase solubility and/or stability and/or glucagon activity.The glucagon peptide may alternatively comprise other modifications thatdo not substantially affect solubility or stability, and that do notsubstantially decrease glucagon activity. In exemplary embodiments, theglucagon peptide may comprise a total of 1, up to 2, up to 3, up to 4,up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acidmodifications relative to the native glucagon sequence.

Exemplary modifications include but are not limited to:

conservative substitutions, for example, conservative substitutions atone or more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19,20, 21, 24, 27, 28 or 29;

modification of the aspartic acid at position 15, for example, bysubstitution with glutamic acid, homoglutamic acid, cysteic acid orhomocysteic acid, which may reduce degradation;

addition of a hydrophilic moiety such as the water soluble polymerpolyethylene glycol, as described herein, e.g. at position 16, 17, 20,21, 24 or 29, which may increase solubility and/or half-life;

modification of the amino acid at position 27, for example, bysubstitution with methionine, leucine or norleucine;

modifications at position 1 or 2 as described herein;

C-terminal extensions as described herein;

homodimerization or heterodimerization as described herein; and

combinations of the above.

In accordance with one embodiment the native glucagon peptide of SEQ IDNO: 1 is modified by the substitution of the native amino acid atposition 28 and/or 29 with a negatively charged amino acid (e.g.,aspartic acid or glutamic acid) and optionally the addition of anegatively charged amino acid (e.g., aspartic acid or glutamic acid) tothe carboxy terminus of the peptide. In an alternative embodiment thenative glucagon peptide of SEQ ID NO: 1 is modified by the substitutionof the native amino acid at position 29 with a positively charged aminoacid (e.g., lysine, arginine or histidine) and optionally the additionof one or two positively charged amino acid (e.g., lysine, arginine orhistidine) on the carboxy terminus of the peptide. In accordance withone embodiment a glucagon analog having improved solubility andstability is provided wherein the analog comprises the amino acidsequence of SEQ ID NO: 34 with the proviso that at least one amino acidsat position, 28, or 29 is substituted with an acidic amino acid and/oran additional acidic amino acid is added at the carboxy terminus of SEQID NO: 34. In one embodiment the acidic amino acids are independentlyselected from the group consisting of Asp, Glu, cysteic acid andhomocysteic acid.

In accordance with one embodiment a glucagon agonist having improvedsolubility and stability is provided wherein the agonist comprises theamino acid sequence of SEQ ID NO: 33, wherein at least one of the aminoacids at positions 27, 28 or 29 is substituted with a non-native aminoacid residue (i.e. at least one amino acid present at position 27, 28 or29 of the analog is an acid amino acid different from the amino acidpresent at the corresponding position in SEQ ID NO: 1). In accordancewith one embodiment a glucagon agonist is provided comprising thesequence of SEQ ID NO: 33 with the proviso that when the amino acid atposition 28 is asparagine and the amino acid at position 29 isthreonine, the peptide further comprises one to two amino acids,independently selected from the group consisting of Lys, Arg, His, Aspor Glu, added to the carboxy terminus of the glucagon peptide.

It has been reported that certain positions of the native glucagonpeptide can be modified while retaining at least some of the activity ofthe parent peptide. Accordingly, applicants anticipate that one or moreof the amino acids located at positions at positions 2, 5, 7, 10, 11,12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27 or 29 of the peptide of SEQID NO: 11 can be substituted with an amino acid different from thatpresent in the native glucagon peptide, and still retain the enhancedpotency, physiological pH stability and biological activity of theparent glucagon peptide. For example, in accordance with one embodimentthe methionine residue present at position 27 of the native peptide ischanged to leucine or norleucine to prevent oxidative degradation of thepeptide.

In one embodiment a glucagon analog of SEQ ID NO: 33 is provided wherein1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 21 or 24 of the analog differ from thecorresponding amino acid of SEQ ID NO: 1. In accordance with anotherembodiment a glucagon analog of SEQ ID NO: 33 is provided wherein 1 to 3amino acids selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16,17, 18, 19, 20, 21 or 24 of the analog differ from the correspondingamino acid of SEQ ID NO: 1. In another embodiment, a glucagon analog ofSEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 34 is provided wherein 1 to 2amino acids selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16,17, 18, 19, 20, 21 or 24 of the analog differ from the correspondingamino acid of SEQ ID NO: 1, and in a further embodiment those one to twodiffering amino acids represent conservative amino acid substitutionsrelative to the amino acid present in the native sequence (SEQ ID NO:1). In one embodiment a glucagon peptide of SEQ ID NO: 11 or SEQ ID NO:13 is provided wherein the glucagon peptide further comprises one, twoor three amino acid substitutions at positions selected from positions2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27 or 29. Inone embodiment the substitutions at positions 2, 5, 7, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 27 or 29 are conservative amino acidsubstitutions.

In one embodiment a glucagon agonist is provided comprising an analogpeptide of SEQ ID NO: 1 wherein the analog differs from SEQ ID NO: 1 byhaving an amino acid other than serine at position 2 and by having anacidic amino acid substituted for the native amino acid at position 28or 29 or an acidic amino acid added to the carboxy terminus of thepeptide of SEQ ID NO: 1. In one embodiment the acidic amino acid isaspartic acid or glutamic acid. In one embodiment a glucagon analog ofSEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 32 is providedwherein the analog differs from the parent molecule by a substitution atposition 2. More particularly, position 2 of the analog peptide issubstituted with an amino acid selected from the group consisting ofd-serine, alanine, glycine, n-methyl serine and amino isobutyric acid.

In another embodiment a glucagon agonist is provided comprising ananalog peptide of SEQ ID NO: 1 wherein the analog differs from SEQ IDNO: 1 by having an amino acid other than histidine at position 1 and byhaving an acidic amino acid substituted for the native amino acid atposition 28 or 29 or an acidic amino acid added to the carboxy terminusof the peptide of SEQ ID NO: 1. In one embodiment the acidic amino acidis aspartic acid or glutamic acid. In one embodiment a glucagon analogof SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 32 isprovided wherein the analog differs from the parent molecule by asubstitution at position 1. More particularly, position 1 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of d-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine and homo-histidine.

In accordance with one embodiment the modified glucagon peptidecomprises a sequence selected from the group consisting of SEQ ID NO: 9,SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 32. In a further embodimenta glucagon peptide is provided comprising a sequence of SEQ ID NO: 9,SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 32 further comprising one totwo amino acids, added to the c-terminus of SEQ ID NO: 9, SEQ ID NO: 12,SEQ ID NO: 13 or SEQ ID NO: 32, wherein the additional amino acids areindependently selected from the group consisting of Lys, Arg, His, AspGlu, cysteic acid or homocysteic acid. In one embodiment the additionalamino acids added to the carboxy terminus are selected from the groupconsisting of Lys, Arg, His, Asp or Glu or in a further embodiment theadditional amino acids are Asp or Glu.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 7 or a glucagon agonist analog thereof. In one embodiment thepeptide comprising a sequence selected from the group consisting of SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.In another embodiment the peptide comprising a sequence selected fromthe group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11.In one embodiment the glucagon peptide comprises the sequence of SEQ IDNO: 8, SEQ ID NO: 10 and SEQ ID NO: 11 further comprising an additionalamino acid, selected from the group consisting of Asp and Glu, added tothe c-terminus of the glucagon peptide. In one embodiment the glucagonpeptide comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 13, and ina further embodiment the glucagon peptide comprises the sequence of SEQID NO: 11.

In accordance with one embodiment a glucagon agonist is providedcomprising a modified glucagon peptide selected from the groupconsisting of:

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R(SEQ ID NO: 34),

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-R(SEQ ID NO: 11) and

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa-Tyr-Leu-Glu-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-R(SEQ ID NO: 13)

wherein Xaa at position 15 is Asp, Glu, cysteic acid, homoglutamic acidor homocysteic acid, the Xaa at position 28 is Asn or an acidic aminoacid and the Xaa at position 29 is Thr or an acidic amino acid and R isan acidic amino acid, COOH or CONH₂, with the proviso that an acidicacid residue is present at one of positions 28, 29 or 30. In oneembodiment R is COOH, and in another embodiment R is CONH₂.

The present disclosure also encompasses glucagon fusion peptides whereina second peptide has been fused to the c-terminus of the glucagonpeptide to enhance the stability and solubility of the glucagon peptide.More particularly, the fusion glucagon peptide may comprise a glucagonagonist analog comprising a glucagon peptideNH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R(SEQ ID NO: 34), wherein R is an acidic amino acid or a bond and anamino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21(KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked to the carboxy terminal aminoacid of the glucagon peptide. In one embodiment the glucagon peptide isselected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 7 or SEQID NO: 8 further comprising an amino acid sequence of SEQ ID NO: 20(GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked tothe carboxy terminal amino acid of the glucagon peptide. In oneembodiment the glucagon fusion peptide comprises SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a glucagon agonistanalog thereof, further comprising an amino acid sequence of SEQ ID NO:20 (GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 (KRNR) linkedto amino acid 29 of the glucagon peptide. In accordance with oneembodiment the fusion peptide further comprises a PEG chain linked to anamino acid at position 16, 17, 21 or 24, wherein the PEG chain isselected from the range of 500 to 40,000 Daltons. In one embodiment theamino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21(KRNRNNIA) or SEQ ID NO: 22 (KRNR) is bound to amino acid 29 of theglucagon peptide through a peptide bond. In one embodiment the glucagonpeptide portion of the glucagon fusion peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 andSEQ ID NO: 13. In one embodiment the glucagon peptide portion of theglucagon fusion peptide comprises the sequence of SEQ ID NO: 11 or SEQID NO: 13, wherein a PEG chain is linked at position 21 or 24,respectively.

In another embodiment the glucagon peptide sequence of the fusionpeptide comprises the sequence of SEQ ID NO: 11, further comprising anamino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21(KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked to amino acid 29 of theglucagon peptide. In one embodiment the glucagon fusion peptidecomprises a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO: 25 and SEQ ID NO: 26. Typically the fusion peptides ofthe present invention will have a c-terminal amino acid with thestandard carboxylic acid group. However, analogs of those sequenceswherein the C-terminal amino acid has an amide substituted for thecarboxylic acid are also encompassed as embodiments. In accordance withone embodiment the fusion glucagon peptide comprises a glucagon agonistanalog selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:11 and SEQ ID NO: 13, further comprising an amino acid sequence of SEQID NO: 23 (GPSSGAPPPS-CONH₂) linked to amino acid 29 of the glucagonpeptide.

The glucagon agonists of the present invention can be further modifiedto improve the peptide's solubility and stability in aqueous solutionswhile retaining the biological activity of the glucagon peptide. Inaccordance with one embodiment, introduction of hydrophilic groups atone or more positions selected from positions 16, 17, 20, 21, 24 and 29of the peptide of SEQ ID NO: 11, or a glucagon agonist analog thereof,are anticipated to improve the solubility and stability of the pHstabilize glucagon analog. More particularly, in one embodiment theglucagon peptide of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, or SEQID NO: 32 is modified to comprise one or more hydrophilic groupscovalently linked to the side chains of amino acids present at positions21 and 24 of the glucagon peptide.

In accordance with one embodiment, the glucagon peptide of SEQ ID NO: 11is modified to contain one or more amino acid substitution at positions16, 17, 20, 21, 24 and/or 29, wherein the native amino acid issubstituted with an amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG. Thenative peptide can be substituted with a naturally occurring amino acidor a synthetic (non-naturally occurring) amino acid. Synthetic ornon-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein.

In one embodiment, a glucagon agonist of SEQ ID NO: 10, SEQ ID NO: 11 orSEQ ID NO: 13 is provided wherein the native glucagon peptide sequencehas been modified to contain a naturally occurring or synthetic aminoacid in at least one of positions 16, 17, 21 and 24 of the nativesequence, wherein the amino acid substitute further comprises ahydrophilic moiety. In one embodiment the substitution is at position 21or 24, and in a further embodiment the hydrophilic moiety is a PEGchain. In one embodiment the glucagon peptide of SEQ ID NO: 11 issubstituted with at least one cysteine residue, wherein the side chainof the cysteine residue is further modified with a thiol reactivereagent, including for example, maleimido, vinyl sulfone, 2-pyridylthio,haloalkyl, and haloacyl. These thiol reactive reagents may containcarboxy, keto, hydroxyl, and ether groups as well as other hydrophilicmoieties such as polyethylene glycol units. In an alternativeembodiment, the native glucagon peptide is substituted with lysine, andthe side chain of the substituting lysine residue is further modifiedusing amine reactive reagents such as active esters (succinimido,anhydride, etc) of carboxylic acids or aldehydes of hydrophilic moietiessuch as polyethylene glycol. IN one embodiment the glucagon peptide isselected fowl the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

In accordance with one embodiment the pegylated glucagon peptidecomprises two or more polyethylene chains covalently bound to theglucagon peptide wherein the total molecular weight of the glucagonchains is about 1,000 to about 5,000 Daltons. In one embodiment thepegylated glucagon agonist comprises a peptide of SEQ ID NO: 6, whereina PEG chain is covalently linked to the amino acid residue at position21 and at position 24, and wherein the combined molecular weight of thetwo PEG chains is about 1,000 to about 5,000 Daltons. In anotherembodiment the pegylated glucagon agonist comprises a peptide of SEQ IDNO: 6, wherein a PEG chain is covalently linked to the amino acidresidue at position 21 and at position 24, and wherein the combinedmolecular weight of the two PEG chains is about 5,000 to about 20,000Daltons.

The polyethylene glycol chain may be in the form of a straight chain orit may be branched. In accordance with one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 500 to about 40,000 Daltons. In one embodiment the polyethyleneglycol chain has a molecular weight selected from the range of about 500to about 5,000 Daltons. In another embodiment the polyethylene glycolchain has a molecular weight of about 20,000 to about 40,000 Daltons.

As described in detail in the Examples, the glucagon agonists of thepresent invention have enhanced biophysical stability and aqueoussolubility in solutions of physiological pH, while retaining ordemonstrating enhanced bioactivity relative to the native peptide.Accordingly, the glucagon agonists of the present invention are believedto be suitable for any use that has previously been described for thenative glucagon peptide. Therefore, the modified glucagon peptidesdescribed herein can be used to treat hypoglycemia, to increase bloodglucose level, to induce temporary paralysis of the gut for radiologicaluses, to reduce and maintain body weight, as adjunctive therapy withinsulin, or to treat other metabolic diseases that result from low bloodlevels of glucagon.

One aspect of the present disclosure is directed to a pre-formulatedaqueous solution of the presently disclosed glucagon agonist for use intreating hypoglycemia. The improved stability and/or solubility of theagonist compositions described herein allow for the preparation ofpre-formulated aqueous solutions of glucagon for rapid administrationand treatment of hypoglycemia. Accordingly, in one embodiment a solutioncomprising a glucagon agonist of the present invention is provided foradministration to a patient suffering from hypoglycemia. In oneembodiment a solution comprising a pegylated glucagon agonist asdisclosed herein is provided for administration to a patient sufferingfrom hypoglycemia, wherein the total molecular weight of the PEG chainslinked to the pegylated glucagon agonist is between about 500 to about5,000 Daltons. In one embodiment the pegylated glucagon agonistcomprises a peptide selected from the group consisting of SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ IDNO: 19, and glucagon agonist analogs of thereof, wherein the side chainof an amino acid residue at position 16, 17, 21 or 24 of said glucagonpeptide is covalently bound to the polyethylene glycol chain. In oneembodiment, the pegylated glucagon agonist comprises the peptide of SEQID NO: 16, wherein the amino acid residue at position 21 of the peptideis covalently linked to polyethylene glycol. In one embodiment, thepegylated glucagon agonist comprises the peptide of SEQ ID NO: 17,wherein the amino acid residue at position 24 of the peptide iscovalently linked to polyethylene glycol.

The method of treating hypoglycemia in accordance with the presentinvention comprises the steps of administering the presently disclosedglucagon agonists to a patient using any standard route ofadministration, including parenterally, such as intravenously,intraperitoneally, subcutaneously or intramuscularly, intrathecally,transdermally, rectally, orally, nasally or by inhalation. In oneembodiment the composition is administered subcutaneously orintramuscularly. In one embodiment, the composition is administeredparenterally and the glucagon composition is prepackaged in a syringe.Advantageously, the aqueous stable glucagon analogs disclosed hereinexhibit superior stability and solubility in aqueous buffers in thebroadest pH range used for pharmacological purposes, relative to nativeglucagon. The use of the stabilized glucagon analogs disclosed hereinallow for the preparation and storage of glucagon agonist solutions atphysiological pH for long periods of time.

Applicants have discovered that pegylated glucagon peptides can beprepared that retain the parent peptide's bioactivity and specificity.However, increasing the length of the PEG chain, or attaching multiplePEG chains to the peptide, such that the total molecular weight of thelinked PEG is greater than 5,000 Daltons, begins to delay the timeaction of the modified glucagon. In accordance with one embodiment, aglucagon peptide of SEQ ID NO: 11 or SEQ ID NO: 13, or a glucagonagonist analog thereof, is provided wherein the peptide comprises one ormore polyethylene glycol chains, wherein the total molecular weight ofthe linked PEG is greater than 5,000 Daltons, and in one embodiment isgreater than 10,000 Daltons, but less than 40,000 Daltons. Such modifiedglucagon peptides have a delayed time of activity but without loss ofthe bioactivity. Accordingly, such compounds can be administeredprophylactically to extend the effect of the administered glucagonpeptide.

Glucagon peptides that have been modified to be covalently bound to aPEG chain having a molecular weight of greater than 10,000 Daltons canbe administered in conjunction with insulin to buffer the actions ofinsulin and help to maintain stable blood glucose levels in diabetics.The modified glucagon peptides of the present disclosure can beco-administered with insulin as a single composition, simultaneouslyadministered as separate solutions, or alternatively, the insulin andthe modified glucagon peptide can be administered at different timerelative to one another. In one embodiment the composition comprisinginsulin and the composition comprising the modified glucagon peptide areadministered within 12 hours of one another. The exact ratio of themodified glucagon peptide relative to the administered insulin will bedependent in part on determining the glucagon levels of the patient, andcan be determined through routine experimentation.

In accordance with one embodiment a composition is provided comprisinginsulin and a modified glucagon peptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 andglucagon agonist analogs thereof, wherein the modified glucagon peptidefurther comprises a polyethylene glycol chain covalently bound to anamino acid side chain at position 16, 17, 21 or 24. In one embodimentthe composition is an aqueous solution comprising insulin and theglucagon analog. In embodiments wherein the glucagon peptide comprisesthe sequence of SEQ ID NO: 11 or SEQ ID NO: 13, the peptide may furthercomprise a polyethylene glycol chain covalently bound to an amino acidside chain at position 16, 17, 21 or 24. In one embodiment the molecularweight of the PEG chain of the modified glucagon peptide is greater than10,000 Daltons. In one embodiment the pegylated glucagon peptidecomprises a peptide selected from the group consisting of SEQ ID NO: 11and SEQ ID NO: 13 wherein the side chain of an amino acid residue atposition 21 or 24 of said glucagon peptide is covalently bound to thepolyethylene glycol chain. In one embodiment the polyethylene glycolchain has a molecular weight of about 10,000 to about 40,000.

In accordance with one embodiment the modified glucagon peptidesdisclosed herein are used to induce temporary paralysis of theintestinal tract. This method has utility for radiological purposes andcomprises the step of administering an effective amount of apharmaceutical composition comprising a pegylated glucagon peptide, aglucagon peptide comprising a c-terminal extension or a dimer of suchpeptides. In one embodiment the glucagon peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. Inone embodiment the glucagon peptide further comprises a PEG chain, ofabout 1,000 to 40,000 Daltons is covalently bound to an amino acidresidue at position 21 or 24. In one embodiment the glucagon peptide isselected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16 and SEQ ID NO: 17. In one embodiment the PEG chain has amolecular weight of about 500 to about 5,000 Daltons.

In a further embodiment the composition used to induce temporaryparalysis of the intestinal tract comprises a first modified glucagonpeptide and a second modified glucagon peptide, wherein the firstmodified peptide comprises a sequence selected from the group consistingof SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 13, optionally linked toa PEG chain of about 500 to about 5,000 Daltons, and the second peptidecomprises a covalently linked PEG chain of about 10,000 to about 40,000Daltons. In this embodiment the PEG chain of each peptide is covalentlybound to an amino acid residue at either position 21 or 24 of therespective peptide, and independent of one another.

Oxyntomodulin, a naturally occurring digestive hormone found in thesmall intestine, has been reported to cause weight loss whenadministered to rats or humans (see Diabetes 2005; 54:2390-2395).Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acidsequence of glucagon (i.e. SEQ ID NO: 1) followed by an 8 amino acidcarboxy terminal extension of SEQ ID NO: 23 (KRNRNNIA). Accordingly,applicants believe that the bioactivity of oxyntomodulin can be retained(i.e. appetite suppression and induced weight loss/weight maintenance),while improving the solubility and stability of the compound andimproving the pharmacokinetics, by substituting the glucagon peptideportion of oxyntomodulin with the modified glucagon peptides disclosedherein. In addition applicants also believe that a truncatedoxyntomodulin molecule comprising a glucagon peptide of the invention,having the terminal four amino acids of oxyntomodulin removed, will alsobe effective in suppressing appetite and inducing weight loss/weightmaintenance.

Accordingly, the present invention also encompasses the modifiedglucagon peptides of the present invention that have a carboxy terminalextension of SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22. In accordancewith one embodiment a glucagon agonist analog of SEQ ID NO: 33, furthercomprising the amino acid sequence of SEQ ID NO: 21 (KRNRNNIA) or SEQ IDNO: 22 linked to amino acid 29 of the glucagon peptide, is administeredto individuals to induce weight loss or prevent weight gain. Inaccordance with one embodiment a glucagon agonist analog of SEQ ID NO:11 or SEQ ID NO: 13, further comprising the amino acid sequence of SEQID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 linked to amino acid 29 of theglucagon peptide, is administered to individuals to induce weight lossor prevent weight gain. In another embodiment a method of reducingweight gain or inducing weight loss in an individual comprisesadministering an effective amount of a composition comprising a glucagonagonist comprising a glucagon peptide selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, whereinamino acid 29 of the glucagon peptide is bound to a second peptidethrough a peptide bond, and said second peptide comprises the sequenceof SEQ ID NO: 24 (KRNRNNIA) or SEQ ID NO: 25, and wherein a PEG chain ofabout 1,000 to 40,000 Daltons is covalently bound to an amino acidresidue at position 21 and/or 24. In one embodiment the glucagon peptidesegment of the glucagon agonist is selected from the group consisting ofSEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18 and SEQ ID NO: 19, wherein a PEG chain of about 1,000 to 40,000Daltons is covalently bound to an amino acid residue at position 16, 17,21 or 24.

Exendin-4, is a peptide made up of 39 amino acids. It is a powerfulstimulator of a receptor known as GLP-1. This peptide has also beenreported to suppress appetite and induce weight loss. Applicants havefound that the terminal sequence of Exendin-4 when added at the carboxyterminus of glucagon improves the solubility and stability of glucagonwithout compromising the bioactivy of glucagon. In one embodiment theterminal ten amino acids of Exendin-4 (i.e. the sequence of SEQ ID NO:20 (GPSSGAPPPS)) are linked to the carboxy terminus of a glucagonpeptide of the present disclosure. These fusion proteins are anticipatedto have pharmacological activity for suppressing appetite and inducingweight loss/weight maintenance. In one embodiment the terminal aminoacid of the SEQ ID NO: 20 extension comprises an amide group in place ofthe carboxy group (i.e., SEQ ID NO: 23) and this sequence is linked tothe carboxy terminus of a glucagon peptide of the present disclosure.

In one embodiment a method of reducing weight gain or inducing weightloss in an individual comprises administering an effective amount of acomposition comprising a glucagon agonist comprising a glucagon peptideof SEQ ID NO: 33 wherein amino acid 29 of the glucagon peptide is boundto a second peptide through a peptide bond, and said second peptidecomprises the sequence of SEQ ID NO: 20 (GPSSGAPPPS) or SEQ ID NO: 23.In one embodiment the glucagon peptide is selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 wherein amino acid 29 ofthe glucagon peptide is bound to a second peptide through a peptidebond, and said second peptide comprises the sequence of SEQ ID NO: 20(GPSSGAPPPS) or SEQ ID NO: 23. In one embodiment the glucagon peptide ofthe glucagon agonist is selected from the group consisting of SEQ ID NO:11 and SEQ ID NO: 13. In one embodiment the glucagon peptide segment ofthe fusion peptide is selected from the group consisting of SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, wherein themolecular weight of the PEG chain is selected from the range of 500 to40,000 Daltons. More particularly, in one embodiment the glucagonpeptide of the fusion peptide is selected from the group consisting ofSEQ ID NO: 16 and SEQ ID NO: 17 wherein the molecular weight of the PEGchain is selected from the range of 1,000 to 5,000.

In another embodiment a composition is administered to a patient tosuppress appetite, prevent weight gain and/or induce weight loss by theadministration of a pharmaceutical composition comprising a firstpegylated glucagon peptide and a second pegylated glucagon peptide,wherein the first and second peptide are fusion peptides comprising ac-terminal peptide extension comprising SEQ ID NO: 20 (GPSSGAPPPS) orSEQ ID NO: 23. The first pegylated glycogen peptide comprising acovalently linked PEG of about 500 to about 10,000 Daltons and thesecond pegylated glucagon peptide comprising a covalently linked PEGchain of about 10,000 to about 40,000 Daltons.

The present disclosure also encompasses multimers of the modifiedglucagon peptides disclosed herein. Two or more of the modified glucagonpeptides can be linked together using standard linking agents andprocedures known to those skilled in the art. For example, dimers can beformed between two modified glucagon peptides through the use ofbifunctional thiol crosslinkers and bi-functional amine crosslinkers,particularly for the glucagon peptides that have been substituted withcysteine, lysine ornithine, homocysteine or acetyl phenylalanineresidues (e.g. SEQ ID NO: 4 and SEQ ID NO: 5). The dimer can be ahomodimer or alternatively can be a heterodimer. In one embodiment thedimer comprises a homodimer of a glucagon fusion peptide wherein theglucagon peptide portion comprises an agonist analog of SEQ ID NO: 11and an amino acid sequence of SEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21(KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked to amino acid 29 of theglucagon peptide. In another embodiment the dimer comprises a homodimerof a glucagon agonist analog of SEQ ID NO: 11, wherein the glucagonpeptide further comprises a polyethylene glycol chain covalently boundto position 21 or 24 of the glucagon peptide.

In accordance with one embodiment a dimer is provided comprising a firstglucagon peptide bound to a second glucagon peptide via a linker,wherein the first glucagon peptide comprises a peptide selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10 and SEQ ID NO: 11 and the second glucagon peptidecomprises SEQ ID NO: 33. Furthermore, with regards to the secondglucagon peptide, when the amino acid at position 28 is asparagine andthe amino acid at position 29 is threonine, the second glucagon peptidefurther comprises one to two amino acids (independently selected fromthe group consisting of Lys, Arg, His, Asp or Glu), added to the carboxyterminus of the second glucagon glucagon peptide, and pharmaceuticallyacceptable salts of said glucagon polypeptides.

In accordance with another embodiment a dimer is provided comprising afirst glucagon peptide bound to a second glucagon peptide via a linker,wherein said first glucagon peptide is selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12and SEQ ID NO: 13 and the second glucagon peptide is independentlyselected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 13,and pharmaceutically acceptable salts of said glucagon polypeptides. Inone embodiment the first glucagon peptide is selected from the groupconsisting of SEQ ID NO: 7 and the second glucagon peptide isindependently selected from the group consisting of SEQ ID NO: 11, SEQID NO: 12 and SEQ ID NO: 13. In one embodiment the dimer is formedbetween two peptides wherein each peptide comprising the amino acidsequence of SEQ ID NO: 11.

In accordance with one embodiment a pharmaceutical composition isprovided wherein the composition comprises a glucagon agonist analog ofthe present disclosure, or pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier. In one embodiment thepharmaceutical composition comprises a 1 mg/ml concentration of theglucagon agonist analog and 10-50 mM Triethanolamine at pH 7.0-8.5. Inone embodiment the pharmaceutical composition comprises a 1 mg/mlconcentration of the glucagon agonist analog and 20 mM Triethanolamineat pH 8.5.

The modified glucagon peptides of the present invention can be providedin accordance with one embodiment as part of a kit. In one embodiment akit for administering a glucagon agonist to a patient in need thereof isprovided wherein the kit comprises any of the glucagon peptides of theinvention in aqueous solution. Exemplary glucagon peptides for inclusionin such kits include a glucagon peptide selected from the groupconsisting of 1) a glucagon peptide comprising the sequence of SEQ IDNO: 7, SEQ ID NO: 10, SEQ ID NO:11 or SEQ ID NO: 13 or SEQ ID NO: 33; 2)a glucagon fusion peptide comprising a glucagon agonist analog of SEQ IDNO: 11 or SEQ ID NO: 13 or SEQ ID NO: 33, and an amino acid sequence ofSEQ ID NO: 20 (GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22(KRNR) linked to amino acid 29 of the glucagon peptide; and 3) apegylated glucagon peptide of SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ IDNO: 33, further comprising an amino acid sequence of SEQ ID NO: 20(GPSSGAPPPS), SEQ ID NO: 21 (KRNRNNIA) or SEQ ID NO: 22 (KRNR) linked toamino acid 29 of the glucagon peptide, wherein the PEG chain covalentlybound to position 16, 17, 21 or 24 has a molecular weight of about 500to about 40,000 Daltons. In one embodiment the kit is provided with adevice for administering the glucagon composition to a patient, e.g.syringe needle, pen device, jet injector or other needle-free injector.The kit may alternatively or in addition include one or more of avariety of containers, e.g., vials, tubes, bottles, single ormulti-chambered pre-filled syringes, cartridges, infusion pumps(external or implantable), jet injectors, pre-filled pen devices and thelike, optionally containing the glucagon peptide. Preferably, the kitswill also include instructions for use. In accordance with oneembodiment the device of the kit is an aerosol dispensing device,wherein the composition is prepackaged within the aerosol device. Inanother embodiment the kit comprises a syringe and a needle, and in oneembodiment the glucagon composition is prepackaged within the syringe.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

EXAMPLES

General Synthesis Protocol

Glucagon analogs were synthesized using HBTU-activated “Fast Boc” singlecoupling starting from 0.2 mmole of Boc Thr(OBzl)Pam resin on a modifiedApplied Biosystem 430 A peptide synthesizer. Boc amino acids and HBTUwere obtained from Midwest Biotech (Fishers, Ind.). Side chainprotecting groups used were: Arg(Tos), Asn(Xan), Asp(OcHex),Cys(pMeBzl), His(Bom), Lys(2Cl—Z), Ser(OBzl), Thr(OBzl), Tyr(2Br—Z), andTrp(CHO). The side-chain protecting group on the N-terminal His was Boc.

Each completed peptidyl resin was treated with a solution of 20%piperidine in dimethylformamide to remove the formyl group from thetryptophan. Liquid hydrogen fluoride cleavages were performed in thepresence of p-cresol and dimethyl sulfide. The cleavage was run for 1hour in an ice bath using an HF apparatus (Pennisula Labs). Afterevaporation of the HF, the residue was suspended in diethyl ether andthe solid materials were filtered. Each peptide was extracted into 30-70ml aqueous acetic acid and a diluted aliquot was analyzed by HPLC[Beckman System Gold, 0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, Abuffer=0.1% TFA, B=0.1% TFA/90% acetonitrile, gradient of 10% to 80% Bover 10 min].

Purification was done on a FPLC over a 2.2×25 cm Kromasil C18 columnwhile monitoring the UV at 214 nm and collecting 5 minute fractions. Thehomogeneous fractions were combined and lyophilized to give a productpurity of >95%. The correct molecular mass and purity were confirmedusing MALDI-mass spectral analysis.

General Pegylation Protocol: (Cys-Maleimido)

Typically, the glucagon Cys analog is dissolved in phosphate bufferedsaline (5-10 mg/ml) and 0.01M ethylenediamine tetraacetic acid is added(10-15% of total volume). Excess (2-fold) maleimido methoxyPEG reagent(Nektar) is added and the reaction stirred at room temp while monitoringreaction progress by HPLC. After 8-24 hrs, the reaction mixture, isacidified and loaded onto a preparative reverse phase column forpurification using 0.1% TFA/acetonitrile gradient. The appropriatefractions were combined and lyophilized to give the desired pegylatedanalogs.

Example 1 Synthesis of Glucagon Cys¹⁷ (1-29) and Similar MonoCys Analogs

0.2 mmole Boc Thr(OBzl) Pam resin (SynChem Inc) in a 60 ml reactionvessel and the following sequence was entered and run on a modifiedApplied Biosystems 430A Peptide Synthesizer using FastBoc HBTU-activatedsingle couplings.

HSQGTFTSDYSKYLDSCRAQDFVQWLMNT (SEQ ID NO: 27)

The following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to an HF reaction vessel anddried in vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were addedalong with a magnetic stir bar. The vessel was attached to the HFapparatus (Pennisula Labs), cooled in a dry ice/methanol bath,evacuated, and aprox. 10 ml liquid hydrogen fluoride was condensed in.The reaction was stirred in an ice bath for 1 hr then the HF was removedin vacuo. The residue was suspended in ethyl ether; the solids werefiltered, washed with ether, and the peptide extracted into 50 mlaqueous acetic acid. An analytical HPLC was run [0.46×5 cm Zorbax C8, 1ml/min, 45 C, 214 nm, A buffer of 0.1% TFA, B buffer of 0.1% TFA/90%ACN, gradient=10% B to 80% B over 10 min.] with a small sample of thecleavage extract. The remaining extract was loaded onto a 2.2×25 cmKromasil C18 preparative reverse phase column and an acetonitrilegradient was run using a Pharmacia FPLC system. 5 min fractions werecollected while monitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1%TFA/50% acetonitrile. Gradient=30% B to 100% B over 450 min.

The fractions containing the purest product (48-52) were combinedfrozen, and lyophilized to give 30.1 mg. An HPLC analysis of the productdemonstrated a purity of >90% and MALDI mass spectral analysisdemonstrated the desired mass of 3429.7. Glucagon Cys²¹, Glucagon Cys²⁴,and Glucagon Cys²⁹ were similarly prepared.

Example 2 Synthesis of Glucagon-Cex and Other C-Terminal ExtendedAnalogs

285 mg (0.2 mmole) methoxybenzhydrylamine resin (Midwest Biotech) wasplaced in a 60 ml reaction vessel and the following sequence was enteredand run on a modified Applied Biosystems 430A peptide synthesizer usingFastBoc HBTU-activated single couplings.

(SEQ ID NO: 28) HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGPSSGAPPPS

The following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to HF reaction vessel and driedin vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were added alongwith a magnetic stir bar. The vessel was attached to the HF apparatus(Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, andaprox. 10 ml liquid hydrogen fluoride was condensed in. The reaction wasstirred in an ice bath for 1 hr then the HF was removed in vacuo. Theresidue was suspended in ethyl ether; the solids were filtered, washedwith ether, and the peptide extracted into 50 ml aqueous acetic acid. Ananalytical HPLC was run [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, Abuffer of 0.1% TFA, B buffer of 0.1% TFA/90% ACN, gradient=10% B to 80%B over 10 min.] on an aliquot of the cleavage extract. The extract wasloaded onto a 2.2×25 cm Kromasil C18 preparative reverse phase columnand an acetonitrile gradient was run for elution using a Pharmacia FPLCsystem. 5 min fractions were collected while monitoring the UV at 214 nm(2.0 A). A=0.1% TFA, B=0.1% TFA/50% acetonitrile. Gradient=30% B to 100%B over 450 min. Fractions 58-65 were combined, frozen and lyophilized togive 198.1 mg.

HPLC analysis of the product showed a purity of greater than 95%. MALDImass spectral analysis showed the presence of the desired theoreticalmass of 4316.7 with the product as a C-terminal amide. Oxyntomodulin andoxyntomodulin-KRNR were similarly prepared as the C-terminal carboxylicacids starting with the appropriately loaded PAM-resin.

Example 3 Glucagon Cys¹⁷ Mal-PEG-5K

15.1 mg of Glucagon Cys¹⁷ (1-29) and 27.3 mg methoxypoly(ethyleneglycol) maleimide avg. M. W. 5000 (mPEG-Mal-5000, NektarTherapeutics) were dissolved in 3.5 ml phosphate buffered saline (PBS)and 0.5 ml 0.01M ethylenediamine tetraacetic acid (EDTA) was added. Thereaction was stirred at room temperature and the progress of thereaction was monitored by HPLC analysis [0.46×5 cm Zorbax C8, 1 ml/min,45 C, 214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to80% B over 10 min.].

After 5 hours, the reaction mixture was loaded onto 2.2×25 cm KromasilC18 preparative reverse phase column. An acetonitrile gradient was runon a Pharmacia FPLC while monitoring the UV wavelength at 214 nm andcollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% acetonitrile,gradient=30% B to 100% B over 450 min. The fractions corresponding tothe product were combined, frozen and lyophilized to give 25.9 mg.

This product was analyzed on HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45 C,214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% Bover 10 min.] which showed a purity of aprox. 90%. MALDI (matrixassisted laser desorption ionization) mass spectral analysis showed abroad mass range (typical of PEG derivatives) of 8700 to 9500. Thisshows an addition to the mass of the starting glucagon peptide (3429) ofapproximately 5,000 a.m.u.

Example 4 Glucagon Cys²¹ Mal-PEG-5K

21.6 mg of Glucagon Cys²¹ (1-29) and 24 mg mPEG-MAL-5000 (NektarTherapeutics) were dissolved in 3.5 ml phosphate buffered saline (PBS)and 0.5 ml 0.01M ethylene diamine tetraacetic acid (EDTA) was added. Thereaction was stirred at room temp. After 2 hrs, another 12.7 mg ofmPEG-MAL-5000 was added. After 8 hrs, the reaction mixture was loadedonto a 2.2×25 cm Vydac C18 preparative reverse phase column and anacetonitrile gradient was run on a Pharmacia FPLC at 4 ml/min whilecollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% ACN. Gradient=20%to 80% B over 450 min.

The fractions corresponding to the appearance of product were combinedfrozen and lyophilized to give 34 mg. Analysis of the product byanalytical HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm (0.5 A),A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10 min.]showed a homogeneous product that was different than starting glucagonpeptide. MALDI (matrix assisted laser desorption ionization) massspectral analysis showed a broad mass range (typical of PEG analogs) of8700 to 9700. This shows an addition to the mass of the startingglucagon peptide (3470) of approximately 5,000 a.m.u.

Example 5 Glucagon Cys²⁴ Mal-PEG-5K

20.1 mg Glucagon C²⁴ (1-29) and 39.5 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring and 0.5 ml0.01M EDTA was added. The reaction was stirred at room temp for 7 hrs,then another 40 mg of mPEG-Mal-5000 was added. After approximately 15hr, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was runusing a Pharmacia FPLC. 5 min. fractions were collected while monitoringthe UV at 214 nm (2.0 A). A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,gradient=30% B to 100% B over 450 min. The fractions corresponding toproduct were combined, frozen and lyophilized to give 45.8 mg. MALDImass spectral analysis showed a typical PEG broad signal with a maximumat 9175.2 which is approximately 5,000 a.m.u. more than Glucagon C²⁴(3457.8).

Example 6 Glucagon Cys²⁴ Mal-PEG-20K

25.7 mg of Glucagon Cys²⁴ (1-29) and 40.7 mg mPEG-Mal-20K (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at room temp.and 0.5 ml 0.01M EDTA was added. After 6 hrs, the ratio of startingmaterial to product was aprox. 60:40 as determined by HPLC. Another 25.1mg of mPEG-Mal-20K was added and the reaction allowed to stir another 16hrs. The product ratio had not significantly improved, so the reactionmixture was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column and purified on a Pharmacia FPLC using a gradient of 30% Bto 100% B over 450 min. A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,flow=4 ml/min, and 5 min fractions were collected while monitoring theUV at 214 nm (2.0 A). The fractions containing homogeneous product werecombined, frozen and lyophilized to give 25.7 mg. Purity as determinedby analytical HPLC was ±90%. A MALDI mass spectral analysis showed abroad peak from 23,000 to 27,000 which is approximately 20,000 a.m.u.more than starting Glucagon C²⁴ (3457.8).

Example 7 Glucagon Cys²⁹ Mal-PEG-5K

20.0 mg of Glucagon Cys²⁹ (1-29) and 24.7 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at roomtemperature and 0.5 ml 0.01M EDTA was added. After 4 hr, another 15.6 mgof mPEG-Mal-5000 was added to drive the reaction to completion. After 8hrs, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was run ona Pharmacia FPLC system. 5 min fractions were collected while monitoringthe UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN. Fractions75-97 were combined frozen and lyophilized to give 40.0 mg of productthat is different than recovered starting material on HPLC (fractions58-63). Analysis of the product by analytical HPLC [0.46×5 cm Zorbax C8,1 ml/min, 45 C, 214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN,gradient=10% B to 80% B over 10 min.] showed a purity greater than 95%.MALDI mass spectral analysis showed the presence of a PEG component witha mass range of 8,000 to 10,000 (maximum at 9025.3) which is 5,540a.m.u. greater than starting material (3484.8).

Example 8 Glucagon Cys²⁴ (2-butyrolactone)

To 24.7 mg of Glucagon Cys²⁴ (1-29) was added 4 ml 0.05M ammoniumbicarbonate/50% acetonitrile and 5.5 ul of a solution of2-bromo-4-hydroxybutyric acid-γ-lactone (100 ul in 900 ul acetonitrile).After 3 hrs of stirring at room temperature, another 105 ul of lactonesolution was added to the reaction mixture which was stirred another 15hrs. The reaction mixture was diluted to 10 ml with 10% aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column. An acetonitrile gradient (20% B to 80% B over 450 min) wasrun on a Pharmacia FPLC while collecting 5 min fractions and monitoringthe UV at 214 nm (2.0 A). Flow=4 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 74-77 were combined frozen and lyophilized to give 7.5 mg.HPLC analysis showed a purity of 95% and MALDI mass spect analysisshowed a mass of 3540.7 or 84 mass units more than starting material.This result consistent with the addition of a single butyrolactonemoiety.

Example 9 Glucagon Cys²⁴ (S-carboxymethyl)

18.1 mg of Glucagon Cys²⁴ (1-29) was dissolved in 9.4 ml 0.1M sodiumphosphate buffer (pH=9.2) and 0.6 ml bromoacetic acid solution (1.3mg/ml in acetonitrile) was added. The reaction was stirred at roomtemperature and the reaction progress was followed by analytical HPLC.After 1 hr another 0.1 ml bromoacetic acid solution was added. Thereaction was stirred another 60 min. then acidified with aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column for purification. An acetonitrile gradient was run on aPharmacia FPLC (flow=4 ml/min) while collecting 5 min fractions andmonitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 26-29 were combined frozen and lyophilized to give several mgof product. Analytical HPLC showed a purity of 90% and MALDI massspectral analysis confirmed a mass of 3515 for the desired product.

Example 10 Glucagon Cys²⁴ maleimido, PEG-3.4K-dimer

16 mg Glucagon Cys²⁴ and 1.02 mg Mal-PEG-Mal-3400,poly(ethyleneglycol)-bis-maleimide avg. M. W. 3400, (NektarTherapeutics) were dissolved in 3.5 phosphate buffered saline and 0.5 ml0.01M EDTA and the reaction was stirred at room temperature. After 16hrs, another 16 mg of Glucagon Cys²⁴ was added and the stirringcontinued. After approximately 40 hrs, the reaction mixture was loadedonto a Pharmcia PepRPC 16/10 column and an acetonitrile gradient was runon a Pharmacia FPLC while collecting 2 min fractions and monitoring theUV at 214 nm (2.0 A). Flow=2 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 69-74 were combined frozen and lyophilized to give 10.4 mg.Analytical HPLC showed a purity of 90% and MALDI mass spectral analysisshows a component in the 9500-11,000 range which is consistent with thedesired dimer.

Example 11 Glucagon Solubility Assays

A solution (1 mg/ml or 3 mg/ml) of glucagon (or an analog) is preparedin 0.01N HCl. 100 ul of stock solution is diluted to 1 ml with 0.01N HCland the UV absorbance (276 nm) is determined. The pH of the remainingstock solution is adjusted to pH7 using 200-250 ul 0.1M Na₂HPO₄ (pH9.2).The solution is allowed to stand overnight at 4° C. then centrifuged.100 ul of supernatant is then diluted to 1 ml with 0.01N HCl, and the UVabsorbance is determined (in duplicate).

The initial absorbance reading is compensated for the increase in volumeand the following calculation is used to establish percent solubility:

${\frac{{Final}\mspace{14mu}{Absorbance}}{{Initial}\mspace{14mu}{Absorbance}} \times 100} = {{percent}\mspace{14mu}{soluble}}$

Results are shown in Table 1 wherein Glucagon-Cex represents wild typeglucagon (SEQ ID NO: 1) plus a carboxy terminal addition of SEQ ID NO:20 and Glucagon-Cex R¹² represents SEQ ID NO: 1 wherein the Lys atposition 12 is substituted with Arg and a peptide of SEQ ID NO: 20 isadded to the carboxy terminus.

TABLE 1 Solubility date for glucagon analogs Analog Percent SolubleGlucagon 16 Glucagon-Cex, R12 104 Glucagon-Cex 87 Oxyntomodulin 104Glucagon, Cys17PEG5K 94 Glucagon, Cys21PEG5K 105 Glucagon, Cys24PEG5K133

The solubility the glucagon agonist analogs, D28, E29, E30, E15D28,D28E30, D28E29, was investigated using the same assay used for thecompounds listed in Table 1. The data (shown in FIGS. 3 & 4)demonstrates the superior solubility of the D28, E29, E30, E15D28,D28E30, D28E29 analogs relative to native glucagon at pH values of 5.5and 7.0. The data presented in FIG. 3 represents the solubility measuredafter 60 hours at 25° C., whereas the data presented in FIG. 4represents the solubility measured after 24 hours at 25° C. and then 24hours at 4° C. FIG. 5 represents data regarding the maximum solubilityof the glucagon analogs D28, D28E30 and E15D28.

Example 12 Glucagon Receptor Binding Assay

The affinity of peptides to the glucagon receptor was measured in acompetition binding assay utilizing scintillation proximity assaytechnology. Serial 3-fold dilutions of the peptides made inscintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 MNaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clearbottom plate (Corning Inc., Acton, Mass.) with 0.05 nM(3-[¹²⁵I]-iodotyrosyl) Tyr 10 glucagon (Amersham Biosciences,Piscataway, N.J.), 1-6 micrograms per well, plasma membrane fragmentsprepared from cells over-expressing human glucagon receptor, and 1mg/well polyethyleneimine-treated wheat germ agglutinin type Ascintillation proximity assay beads (Amersham Biosciences, Piscataway,N.J.). Upon 5 min shaking at 800 rpm on a rotary shaker, the plate wasincubated 12 h at room temperature and then read on MicroBeta 1450liquid scintillation counter (Perkin-Elmer, Wellesley, Mass.).Non-specifically bound (NSB) radioactivity was measured in the wellswith 4 times greater concentration of “cold” native ligand than thehighest concentration in test samples and total bound radioactivity wasdetected in the wells with no competitor. Percent specific binding wascalculated as following: % Specific Binding=((Bound-NSB)/(Totalbound-NSB))×100. IC₅₀ values were determined by using Origin software(OriginLab, Northampton, Mass.).

Example 13 Functional Assay-cAMP Synthesis

The ability of glucagon analogs to induce cAMP was measured in a fireflyluciferase-based reporter assay. HEK293 cells co-transfected with eitherglucagon- or GLP-1 receptor and luciferase gene linked to cAMPresponsive element were serum deprived by culturing 16 h in DMEM(Invitrogen, Carlsbad, Calif.) supplemented with 0.25% Bovine GrowthSerum (HyClone, Logan, Utah) and then incubated with serial dilutions ofeither glucagon, GLP-1 or novel glucagon analogs for 5 h at 37° C., 5%CO₂ in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation 100 microliters ofLucLite luminescence substrate reagent (Perkin-Elmer, Wellesley, Mass.)were added to each well. The plate was shaken briefly, incubated 10 minin the dark and light output was measured on MicroBeta-1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). Effective 50%concentrations were calculated by using Origin software (OriginLab,Northampton, Mass. Results are shown in Tables 2 and 3.

TABLE 2 cAMP Induction by Glucagon Analogs with C-Terminus ExtensioncAMP Induction Glucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N*EC₅₀, nM N Glucagon 0.22 ± 0.09 14 3.85 ± 1.64 10 GLP-1 2214.00 ±182.43  2 0.04 ± 0.01 14 Glucagon Cex 0.25 ± 0.15 6 2.75 ± 2.03 7Oxyntomodulin 3.25 ± 1.65 5 2.53 ± 1.74 5 Oxyntomodulin 2.77 ± 1.74 43.21 ± 0.49 2 KRNR Glucagon R12 0.41 ± 0.17 6 0.48 ± 0.11 5 Glucagon R12Cex 0.35 ± 0.23 10 1.25 ± 0.63 10 Glucagon R12 K20 0.84 ± 0.40 5 0.82 ±0.49 5 Glucagon R12 K24 1.00 ± 0.39 4 1.25 ± 0.97 5 Glucagon R12 K290.81 ± 0.49 5 0.41 ± 0.24 6 Glucagon Amide 0.26 ± 0.15 3 1.90 ± 0.35 2Oxyntomodulin C24 2.54 ± 0.63 2 5.27 ± 0.26 2 Oxyntomodulin C24 0.97 ±0.04 1 1.29 ± 0.11 1 PEG 20K *number of experiments

TABLE 3 cAMP Induction by Pegylated Glucagon Analogs cAMP InductionGlucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N* EC₅₀, nM N Glucagon0.33 ± 0.23 18 12.71 ± 3.74 2 Glucagon C17 PEG 5K 0.82 ± 0.15 4 55.86 ±1.13 2 Glucagon C21 PEG 5K 0.37 ± 0.16 6 11.52 ± 3.68 2 Glucagon C24 PEG5K 0.22 ± 0.10 12 13.65 ± 2.95 4 Glucagon C29 PEG 5K 0.96 ± 0.07 2 12.71± 3.74 2 Glucagon C24 PEG 20K 0.08 ± 0.05 3 Not determined Glucagon C24Dimer 0.10 ± 0.05 3 Not determined GLP-1 >1000  0.05 ± 0.02 4 *number ofexperiments

Data for additional glucagon analogs is presented in FIGS. 6-9 and inTable 4

TABLE 4 Observed EC50s (nM) in Cells Overexpressing the GlucagonReceptor Test 1 Test 2 Test 3 Test 4 Glucagon standard 0.12 0.04 0.050.11 K29 0.35 0.22 K30 0.22 0.06 K29, K30 0.89 K30, K31 0.12 D28 0.050.17 E28 0.14 E29 0.05 0.04 E30 0.04 D28, E29 0.03 D28, E30 0.05 D28,E15 0.15

Example 14 Stability Assay for Glucagon Cys-Maleimido PEG Analogs

Each glucagon analog was dissolved in water or PBS and an initial HPLCanalysis was conducted. After adjusting the pH (4, 5, 6, 7), the sampleswere incubated over a specified time period at 37° C. and re-analyzed byHPLC to determine the integrity of the peptide. The concentration of thespecific peptide of interest was determined and the percent remainingintact was calculated relative to the initial analysis. Results forGlucagon Cys²¹-maleimidoPEG_(5K) are shown in FIGS. 1 and 2.

Example 15 Changes in Serum Glucose Concentration in Beagle Dogs afterAdministration of Glucagon Analogs

Canine/Beagle dogs of 8-12 kg, being of 8-16 months of age and goodhealth were used to determine the pharmacokinetics and pharmacodynamicsof glucagon action. Every animal was fasted overnight and bled at thefollowing time points after each dose: 0 hr. (pre-dose), 5, 10, 20, 30,45, 60, 90, 120, 240 minutes post dose. Six animals were used for eachdose group and approximately 1-2 ml of whole blood was withdrawn at eachtime point. About 1.0 ml whole blood was added to K₂ EDTA tubescontaining a sufficient volume of Trasylol (aprotinin) to yield at least500 KIU/mL of whole blood. Approximately 500 uL plasma was collectedafter centrifuging the samples in a refrigerated centrifuge at about1,500-3,000×g for 10-15 min. The samples were transferred to plasticvials and stored frozen at −70° C., or below. The remaining 1.0 mL ofwhole blood was converted into serum by placing blood sample into anempty tube, letting sit at ambient temperature for 15-20 min, thencentrifuging at 1,500-3,000×g for 10-15 min. in a refrigeratedcentrifuge. The samples were transferred to plastic vials and storedfrozen at −70° C., or below. Glucagon and the analogs were dissolved in0.01N HCl at a concentration of 0.1667 mg/ml and the animals were dosedat 0.03 ml/kg.

The animals were administered a 0.005 mg/kg dose intramuscularly ofeither glucagon, a glucagon analog comprising glucagon with the sequenceof SEQ ID NO: 31 linked to the carboxy terminus of glucagon(glucagon-CEX) or a glucagon analog comprising an aspartic acidsubstitution at amino acid 28 (glucagon-Asp28) SEQ ID NO: 11. Theresulting data is presented in FIG. 10.

The invention claimed is:
 1. A glucagon peptide with improved solubilityrelative to native glucagon (SEQ ID NO: 1), that is an analog of SEQ IDNO: 1 comprising (a) one, two, or three charged amino acid(s) C-terminalto the amino acid at position 27 of the glucagon peptide, (b) a Leu orNle at position 27, and (c) up to 4 additional amino acid modificationsrelative to SEQ ID NO: 1, wherein the glucagon peptide retains improvedsolubility and glucagon activity.
 2. The glucagon peptide of claim 1,wherein said one, two, or three charged amino acid are negativelycharged amino acids.
 3. The glucagon peptide of claim 2, wherein thenegatively charged amino acids are selected from the group consisting ofGlu and Asp.
 4. The glucagon peptide of claim 3, wherein (i) the aminoacid at position 28 is Asp, or (ii) the amino acid at position 29 isGlu, or both (i) and (ii).
 5. The glucagon peptide of claim 4, whereinthe additional amino acid modifications are: amino acid substitutions atpositions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, and 24relative to SEQ ID NO:
 1. 6. A homo dimer comprising two glucagonpeptides of claim 5 bound to one another through a linker.
 7. Theglucagon peptide of claim 2, wherein two or more charged amino acidsC-terminal to the amino acid at position 27 are negatively charged. 8.The glucagon peptide of claim 7, comprising a. a substitution of theamino acid at position 15 with Glu, homo-Glu, cysteic acid, homo cysteicacid; b. a hydrophilic moiety covalently bound at position 16, 17, 20,21, 24 or 29 of said glucagon peptide; c. a sequence selected from thegroup consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22bound to the carboxy terminus of said glucagon peptide; d. an amino acidat position 2 selected from the group consisting of d-serine, alanine,glycine, n-methyl serine and amino isobutyric acid; or the amino acid atposition 1 is selected from the group consisting of dhistidine,desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine; or e. a combination thereof.
 9. A glucagon peptide withimproved solubility relative to native glucagon (SEQ ID NO: 1)comprising the sequence of SEQ ID NO: 33 or an analog of SEQ ID NO: 33,wherein said analog differs from SEQ ID NO: 33 by one to three aminoacid substitutions, said amino acid substitutions located at one or morepositions selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16,17, 18, 19, 20, 21 and 24 of the analog, with the proviso that saidglucagon peptide comprises a charged amino acid at position 28 or 29, oran additional acidic amino acid added to the carboxy terminus of thepeptide.
 10. The glucagon peptide of claim 1 wherein the glucagonpeptide comprises the sequence of any one of SEQ ID NOs: 7, 8, 9, 11,12, 13, 32, 33, 34, or
 38. 11. A pharmaceutical composition comprisingthe glucagon peptide of claim 5, 9, or 7 or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier. 12.The glucagon peptide of claim 9 further comprising a hydrophilic moietycovalently bound at position 16, 17, 21 or 24 of the glucagon peptide.13. The glucagon peptide of claim 12 wherein the hydrophilic moiety ispolyethylene glycol (PEG).
 14. The glucagon peptide of claim 13 whereinthe glucagon peptide further comprises a sequence selected from thegroup consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22bound to the carboxy terminus of said glucagon peptide.
 15. A solutioncomprising the glucagon peptide of claim 1, 5, or 9 or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier; said solution at pH of about 5.5 to about 8.0.
 16. Akit for administering a glucagon agonist to a patient in need thereof,said kit comprising the solution of claim 15; and a device foradministering said composition to a patient.
 17. The kit of claim 16wherein said device: is an aerosol dispensing device, wherein thecomposition is prepackaged within the aerosol device; or comprises asyringe and a needle, wherein the composition is prepackaged within thesyringe.
 18. A method of treating hypoglycemia in a patient, comprisingadministering to the patient the glucagon peptide of claim 1, 5, or 9 inan amount effective for treating hypoglycemia.